#METABOLOMICS WORKBENCH AbhaySatoskar_20231017_004228 DATATRACK_ID:4405 STUDY_ID:ST002974 ANALYSIS_ID:AN004883 PROJECT_ID:PR001851
VERSION 1
CREATED_ON November 14, 2023, 1:32 am
#PROJECT
PR:PROJECT_TITLE Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In
PR:PROJECT_TITLE Cutaneous Lesions
PR:PROJECT_TYPE Untargeted MS
PR:PROJECT_SUMMARY Cutaneous leishmaniasis is characterized by extensive skin lesions, which are
PR:PROJECT_SUMMARY usually painless despite being associated with extensive inflammation. The
PR:PROJECT_SUMMARY molecular mechanisms responsible for this analgesia have not been identified.
PR:PROJECT_SUMMARY Through untargeted metabolomics, we found enriched anti-nociceptive metabolic
PR:PROJECT_SUMMARY pathways in L. mexicana-infected mice. Purines were elevated in infected
PR:PROJECT_SUMMARY macrophages and at the lesion site during chronic infection. These purines have
PR:PROJECT_SUMMARY anti-inflammatory and analgesic properties by acting through adenosine
PR:PROJECT_SUMMARY receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also
PR:PROJECT_SUMMARY found arachidonic acid metabolism enriched in the ear lesions compared to the
PR:PROJECT_SUMMARY non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and
PR:PROJECT_SUMMARY 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid
PR:PROJECT_SUMMARY receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic
PR:PROJECT_SUMMARY effects. Our study provides evidence of metabolic pathways upregulated during L.
PR:PROJECT_SUMMARY mexicana infection that may mediate anti-nociceptive effects experienced by CL
PR:PROJECT_SUMMARY patients and identifies macrophages as a source of these metabolites.
PR:INSTITUTE The Ohio State University
PR:DEPARTMENT Pathology and Microbiology
PR:LAST_NAME Satoskar
PR:FIRST_NAME Abhay
PR:ADDRESS Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA
PR:EMAIL abhay.satoskar@osumc.edu
PR:PHONE (614) 293-0537
#STUDY
ST:STUDY_TITLE Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In
ST:STUDY_TITLE Cutaneous Lesions
ST:STUDY_TYPE Untargeted MS
ST:STUDY_SUMMARY Cutaneous leishmaniasis is characterized by extensive skin lesions, which are
ST:STUDY_SUMMARY usually painless despite being associated with extensive inflammation. The
ST:STUDY_SUMMARY molecular mechanisms responsible for this analgesia have not been identified.
ST:STUDY_SUMMARY Through untargeted metabolomics, we found enriched anti-nociceptive metabolic
ST:STUDY_SUMMARY pathways in L. mexicana-infected mice. Purines were elevated in infected
ST:STUDY_SUMMARY macrophages and at the lesion site during chronic infection. These purines have
ST:STUDY_SUMMARY anti-inflammatory and analgesic properties by acting through adenosine
ST:STUDY_SUMMARY receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also
ST:STUDY_SUMMARY found arachidonic acid metabolism enriched in the ear lesions compared to the
ST:STUDY_SUMMARY non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and
ST:STUDY_SUMMARY 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid
ST:STUDY_SUMMARY receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic
ST:STUDY_SUMMARY effects. Our study provides evidence of metabolic pathways upregulated during L.
ST:STUDY_SUMMARY mexicana infection that may mediate anti-nociceptive effects experienced by CL
ST:STUDY_SUMMARY patients and identifies macrophages as a source of these metabolites.
ST:INSTITUTE The Ohio State University
ST:DEPARTMENT Pathology and Microbiology
ST:LAST_NAME Satoskar
ST:FIRST_NAME Abhay
ST:ADDRESS Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA
ST:EMAIL abhay.satoskar@osumc.edu
ST:PHONE (614) 293-0537
#SUBJECT
SU:SUBJECT_TYPE Mammal
SU:SUBJECT_SPECIES Mus musculus
SU:TAXONOMY_ID 10090
SU:GENDER Female
#FACTORS
#SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control1_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control2_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_1 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_2 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-Control3_3 Sample Type:Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex1_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex2_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_1 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_2 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMSup-LMex3_3 Sample Type:Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control1_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control2_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_1 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_2 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-Control3_3 Sample Type:Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex1_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex2_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_1 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_2 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - BMDMPellet-LMex3_3 Sample Type:Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_1 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_2 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N1_3 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_1 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_2 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_N2_3 Sample Type:Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi1_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi2_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi3_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_1 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 1 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_2 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 2 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - VolpedoTissue_Ipsi4_3 Sample Type:Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 3 Mass spec mode=Positive mode & Negative mode
SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L1_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L2_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_1 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_2 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-L3_Cell_3 Sample Type:Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 3 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_1 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 1 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_2 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 2 Mass spec mode=Positive
SUBJECT_SAMPLE_FACTORS - RAW-N_Cell_3 Sample Type:Cell pellet derived from naïve RAW 264.7. Technical replicate 3 Mass spec mode=Positive
#COLLECTION
CO:COLLECTION_SUMMARY For in vivo studies, the ears were collected, snap frozen, and processed for
CO:COLLECTION_SUMMARY mass spectrometry analysis. For in vitro experiments, the culture supernatant
CO:COLLECTION_SUMMARY was collected and cell debris was removed by centrifugation. The attached cells
CO:COLLECTION_SUMMARY were be scraped, washed with PBS and quenched with 80% methanol. Then they were
CO:COLLECTION_SUMMARY snap-frozen, centrifuged, and lyophilized
CO:COLLECTION_PROTOCOL_FILENAME Study_Methods-Volpedo_et_al.pdf
CO:SAMPLE_TYPE Biopsy
CO:STORAGE_CONDITIONS -80℃
#TREATMENT
TR:TREATMENT_SUMMARY Experimental mice were infected intradermally in the ear with 1 million L.
TR:TREATMENT_SUMMARY mexicana promastigotes. Naive mice received PBS injection. RAW 264.7 macrophages
TR:TREATMENT_SUMMARY and BMDMs were plated in a 24-well plate at a density of 0.5x106 per well and
TR:TREATMENT_SUMMARY infected overnight with stationary phase L. mexicana promastigotes at a ratio of
TR:TREATMENT_SUMMARY 10:1 (parasite to macrophages). The controls were treated with media alone.
TR:TREATMENT_SUMMARY Then, the extracellular parasites were removed by washing with PBS and new media
TR:TREATMENT_SUMMARY was applied. After a 24hrs incubation, the supernatant and cell pellet were
TR:TREATMENT_SUMMARY collected and processed for mass spectrometry.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY For in vitro experiments, the culture supernatant was collected and cell debris
SP:SAMPLEPREP_SUMMARY was removed by centrifugation according to SOP 5 of the Laboratory Guide for
SP:SAMPLEPREP_SUMMARY Metabolomics Experiments. The attached cells were be scraped, washed with PBS
SP:SAMPLEPREP_SUMMARY and quenched with 80% methanol. Then they were snap-frozen, centrifuged, and
SP:SAMPLEPREP_SUMMARY lyophilized according to SOP 4 of the Laboratory Guide for Metabolomics
SP:SAMPLEPREP_SUMMARY Experiments. For in vivo studies, the ears were collected, snap frozen, and
SP:SAMPLEPREP_SUMMARY processed for mass spectrometry analysis according to SOP 7 of the Laboratory
SP:SAMPLEPREP_SUMMARY Guide for Metabolomics Experiments. Samples were then incubated with 500 uL of
SP:SAMPLEPREP_SUMMARY 100% MeOH and sonicated. The tissue was weighed and homogenized at 40 mg/mL of
SP:SAMPLEPREP_SUMMARY 50% MeOH solution for 3 cycles in a Precellys homogenizer. The supernatant was
SP:SAMPLEPREP_SUMMARY collected, dried down, and reconstituted in ½ of the original volume in 5 %
SP:SAMPLEPREP_SUMMARY MeOH with 0.1 % formic acid. For metabolite candidates found via untargeted
SP:SAMPLEPREP_SUMMARY analysis, pure standards purchased from Sigma-Aldrich were diluted in 100 % MeOH
SP:SAMPLEPREP_SUMMARY stocks to 10 ug/mL, dried down, and reconstituted in 5 % MeOH with 0.1 % formic
SP:SAMPLEPREP_SUMMARY acid and run in the same conditions described below to match features for
SP:SAMPLEPREP_SUMMARY identification. Untargeted analysis was performed on a Thermo Scientific
SP:SAMPLEPREP_SUMMARY Q-Exactive Plus Orbitrap mass spectrometer (MS) with HPLC separation on a
SP:SAMPLEPREP_SUMMARY Poroshell 120 SB-C18 (2 x 100 mm, 2.7 µm particle size) with a Thermo
SP:SAMPLEPREP_SUMMARY Scientific Ultimate WPS 3000 UHPLC system. The gradient consisted of solvent A,
SP:SAMPLEPREP_SUMMARY H2O with 0.1% Formic acid, and solvent B 100% acetonitrile at a 200 µL/min flow
SP:SAMPLEPREP_SUMMARY rate with an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding
SP:SAMPLEPREP_SUMMARY at 95% B for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B
SP:SAMPLEPREP_SUMMARY until min 30. For each sample, 5 µL was injected for each sample with
SP:SAMPLEPREP_SUMMARY ionization in the MS on a HESI electrospray ionization source using a capillary
SP:SAMPLEPREP_SUMMARY voltage of 4.5 kV in positive and 4.0 kV in negative mode at 320 °C capillary
SP:SAMPLEPREP_SUMMARY temperature and 100 °C probe temperature. Gas settings were set to 15 for
SP:SAMPLEPREP_SUMMARY sheath gas and 5 auxiliary gas while the S-Lens was set to 50 V. The top 5 ions
SP:SAMPLEPREP_SUMMARY were selected for data dependent analysis with a 10 second exclusion window,
SP:SAMPLEPREP_SUMMARY with a mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and
SP:SAMPLEPREP_SUMMARY 17,5000 for MSMS scans and fragmentation normalized collision energies of 30 V.
SP:SAMPLEPREP_SUMMARY For feature selection in the untargeted results analysis, including database
SP:SAMPLEPREP_SUMMARY comparison and statistical processing, samples were analyzed in Progenesis QI
SP:SAMPLEPREP_SUMMARY and the pooled sample runs were selected for feature alignment. Using the pooled
SP:SAMPLEPREP_SUMMARY QC samples, features with above 30% CV and max abundances below 5000 intensity
SP:SAMPLEPREP_SUMMARY were filtered out and Anova p-value scores between the groups were calculated
SP:SAMPLEPREP_SUMMARY with a cutoff of &lt; 0.05. With database matching using the Human Metabolome
SP:SAMPLEPREP_SUMMARY Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and
SP:SAMPLEPREP_SUMMARY M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were
SP:SAMPLEPREP_SUMMARY tentatively identified as potential metabolites. Metabolites were only annotated
SP:SAMPLEPREP_SUMMARY with MSMS fragmentation matching scores above 20 % with Progenesis Metascope.
SP:SAMPLEPREP_PROTOCOL_FILENAME Study_Methods-Volpedo_et_al.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY Untargeted analysis was performed on a Thermo Scientific Q-Exactive Plus
CH:CHROMATOGRAPHY_SUMMARY Orbitrap mass spectrometer (MS) with HPLC separation on a Poroshell 120 SB-C18
CH:CHROMATOGRAPHY_SUMMARY (2 x 100 mm, 2.7 µm particle size) with a Thermo Scientific Ultimate WPS 3000
CH:CHROMATOGRAPHY_SUMMARY UHPLC system. The gradient consisted of solvent A, H2O with 0.1% Formic acid,
CH:CHROMATOGRAPHY_SUMMARY and solvent B 100% acetonitrile at a 200 µL/min flow rate with an initial 2%
CH:CHROMATOGRAPHY_SUMMARY solvent B with a linear ramp to 95% B at 15 min, holding at 95% B for 1 minutes,
CH:CHROMATOGRAPHY_SUMMARY and back to 5% B from 17 min and equilibration of 5% B until min 30. For each
CH:CHROMATOGRAPHY_SUMMARY sample, 5 µL was injected for each sample with ionization in the MS on a HESI
CH:CHROMATOGRAPHY_SUMMARY electrospray ionization source using a capillary voltage of 4.5 kV in positive
CH:CHROMATOGRAPHY_SUMMARY and 4.0 kV in negative mode at 320 °C capillary temperature and 100 °C probe
CH:CHROMATOGRAPHY_SUMMARY temperature. Gas settings were set to 15 for sheath gas and 5 auxiliary gas
CH:CHROMATOGRAPHY_SUMMARY while the S-Lens was set to 50 V.
CH:CHROMATOGRAPHY_TYPE Reversed phase
CH:INSTRUMENT_NAME Thermo Scientific Ultimate WPS 3000 UHPLC system
CH:COLUMN_NAME Agilent Infinity Poroshell 120 SB-C18 (100 x 2 mm,2.7 µm)
CH:SOLVENT_A H2O with 0.1% Formic acid
CH:SOLVENT_B 100% acetonitrile
CH:FLOW_GRADIENT an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding at 95% B
CH:FLOW_GRADIENT for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B until min
CH:FLOW_GRADIENT 30
CH:FLOW_RATE 200 µL/min flow rate
CH:COLUMN_TEMPERATURE 320 °C capillary temperature and 100 °C probe temperature
CH:METHODS_FILENAME Study_Methods-Volpedo_et_al.pdf
#ANALYSIS
AN:ANALYSIS_TYPE MS
AN:ANALYSIS_PROTOCOL_FILE Study_Methods-Volpedo_et_al.pdf
#MS
MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap
MS:INSTRUMENT_TYPE Orbitrap
MS:MS_TYPE ESI
MS:ION_MODE POSITIVE
MS:MS_COMMENTS See Study Methods for full procedures. After LC-MS run, the top 5 ions were
MS:MS_COMMENTS selected for data dependent analysis with a 10 second exclusion window, with a
MS:MS_COMMENTS mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and 17,5000
MS:MS_COMMENTS for MSMS scans and fragmentation normalized collision energies of 30 V. For
MS:MS_COMMENTS feature selection in the untargeted results analysis, including database
MS:MS_COMMENTS comparison and statistical processing, samples were analyzed in Progenesis QI
MS:MS_COMMENTS and the pooled sample runs were selected for feature alignment. Using the pooled
MS:MS_COMMENTS QC samples, features with above 30% CV and max abundances below 5000 intensity
MS:MS_COMMENTS were filtered out and Anova p-value scores between the groups were calculated
MS:MS_COMMENTS with a cutoff of &lt; 0.05. With database matching using the Human Metabolome
MS:MS_COMMENTS Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and
MS:MS_COMMENTS M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were
MS:MS_COMMENTS tentatively identified as potential metabolites. Metabolites were only annotated
MS:MS_COMMENTS with MSMS fragmentation matching scores above 20 % with Progenesis Metascope.
MS:MS_RESULTS_FILE ST002974_AN004883_Results.txt UNITS:ppm Has m/z:Yes Has RT:Yes RT units:Minutes
#END